The goal of the project is to elucidate the molecular mechanisms by which vascular endothelium is damaged during inflammatory reactions. The major focus during the past year has been on endothelial cell damage induced by oxidative species released from phagocytic cells or produced by free hemoglobin in solution. We have developed, in collaboration with Dr. Alayash, an in vitro model system for assessing hemoglobin-induced damage of bovine aorta endothelial cells (BAEC), the role of reactive oxygen species as mediators of damage, and the relative contribution of necrotic and apoptotic cell death pathways. Using this system we demonstrated that the pseudo-peroxidase activity of hemoglobin protected against hydrogen peroxide induced necrosis. Cross-linked hemoglobin derivatives were shown to undergo oxidative damage and to undergo a transition to a stable ferryl (Fe+4) form in the presence of hydrogen peroxide. The isolated ferryl-hemoglobin derivatives were toxic to BAEC and induced apoptotic cell death. Future studies will address the mechanism by which ferryl-hemoglobins induce cell death. In addition to studying direct oxidative damage of endothelial cells, we have continued to examine the role of anti-endothelial cell antibodies in mediating vascular damage in auto-immune diseases. In a variety of vasculitic diseases and in systemic lupus erythematosus (SLE), anti-endothelial cell antibodies are present in patient serum and are hypothesized to contribute to disease pathogenesis. Little is known, however, about the mechanisms by which they contribute to coronary artery disease and thrombosis. Binding of antibodies specifically to apoptotic endothelial cells could result in opsonized particles capable of initiating macrophage activation and release of pro-inflammatory mediators. To test this hypothesis, apoptotic human umbilical vein endothelial cells (HUVEC) were purified after serum-deprivation and used to characterize anti-apoptotic endothelial cell antibodies found in sera obtained from SLE patients enrolled in the Hopkins Lupus Cohort (collaboration with Dr. Michelle Petri). A subset of SLE patients expressed antibodies to apoptotic HUVEC. These antibodies could be classified as either anti-endothelial cell antibodies (which also bind to healthy HUVEC) or as anti-cardiolipin antibodies (which bind to negatively charged phospholipids such as cardiolipin, but not to healthy HUVEC). Future studies will address whether antibodies bound to apoptotic HUVEC can elicit an inflammatory response from phagocytic cells.